The fields of -omics, which started to be studied after the completion of the Human Genome Project, systemically deal with qualitative/quantitative information such as nucleic acids, proteins, carbohydrates and fats and are the most actively researched analysis chemical fields. Representative omics are proteomics involving protein analysis and metabolomics involving metabolites. In these fields, a variety of methods may be used to analyze peptides or low-molecular weight substances. Spectroscopic analysis such as nuclear magnetic resonance, infrared spectroscopy and Raman spectroscopy, as well as mass spectrometry in which masses of substances are measured after ionization by various methods may be used.
Of them, regarding mass spectrometry, analytical technology such as electrophoresis, chromatography, or ultracentrifugation should be used to measure the mass of biopolymers such as proteins till the late 1970's. However, these methods have considerably low accuracy of analysis results because many analysis errors are generated by factors such as shape, hydrophilic groups and hydrophobic groups and dissociation levels of biopolymers when fluids flow.
Accordingly, two new ionization technologies (ESI and MALDI) to overcome these difficulties were introduced in the early of 1990s, thus realizing mass and structure analysis of proteins and mass spectrometry starts to play a great role in the proteomics, the study field thereof.
In particular, on-line solid phase extraction/capillary reverse-phase liquid chromatography is considered very important in proteome research due to excellent analysis efficiency. On-line solid phase extraction/capillary reverse-phase liquid chromatography enables effective analysis of fine amounts of biomaterials and identification of fine amounts of proteins at high efficiency due to wide analyte-solid reaction range.
As a method of analyzing proteins, mass spectrometry-based methods function as a standard analysis platform for proteome research. Representative examples of mass spectrometry-based methods include shotgun, bottom-up methods and the like which involve degradation of proteins into peptides by hydrolysis before analysis using a mass spectrometer. This hydrolysis causes formation of peptide fragments which can be easily ionized and detected in a mass spectrometer while increasing solubility of bio-samples. However, this process inevitably causes complexity of samples. For example, in the simplest proteome, the yeast proteome, 300,000 or more peptide fragments are produced from about 6,000 various proteins.
Accordingly, in an attempt to solve this sample complexity, a variety of methods such as on-/off-line multidimensional protein identification technology described in Non-patent document 1 were developed, but the need for improvement in efficiency and sensitivity of liquid chromatography columns still remains.
In this case, it was known that the sensitivity of liquid chromatography/mass spectrometry tests can be rapidly increased when the inner diameter of separation columns is decreased while maintaining a predetermined length of separation columns, as described in Non-patent document 2.
However, conventional on-line reverse-phase liquid chromatography devices have a problem of taking a long time for column equilibration or re-use, when capillary columns having a large length and a small inner diameter are filled with a hydrophobic medium. For example, to re-use a column with a length of 1 m and an inner diameter of 75 μm, at least two hours are required for equilibration.
Accordingly, conventional on-line reverse-phase liquid chromatography devices have problems of considerable loss in terms of cost and time, due to dead time at which other analysis processes cannot be conducted during cleaning and equilibration or regeneration of columns.